The invention relates to recombinant DNA technology. In particular, the invention relates to compositions, including vectors, and methods for the rapid subcloning of nucleic acid sequences in vivo and in vitro.
Molecular biotechnology has revolutionized the production of protein and polypeptide compounds of pharmacological importance. The advent of recombinant DNA technology permitted for the first time the production of proteins on a large scale in a recombinant host cell rather than by the laborious and expensive isolation of the protein from tissues which may only contain minute quantities of the desired protein (e.g., isolation of human growth hormone from cadaver pituitary). The production of proteins, including human proteins, on a large scale in a heterologous host requires the ability to express the protein of interest in the heterologous host. This process typically involves isolation or cloning of the gene encoding the protein of interest followed by transfer of the coding region into an expression vector that contains elements (e.g., promoters) which direct the expression of the desired protein in the heterologous host cell. The most commonly used means of transferring or subcloning a coding region into an expression vector involves the in vitro use of restriction endonucleases and DNA ligases. Restriction endonucleases are enzymes which generally recognize and cleave a specific DNA sequence in a double-stranded DNA molecule. Restriction enzymes are used to excise the coding region from the cloning vector and the excised DNA fragment is then joined using DNA ligase to a suitably cleaved expression vector in such a manner that a functional protein may be expressed.
The ability to transfer the desired coding region to an expression vector is often limited by the availability or suitability of restriction enzyme recognition sites. Often multiple restriction enzymes must be employed for the removal of the desired coding region and the reaction conditions used for each enzyme may differ such that it is necessary to perform the excision reactions in separate steps. In addition, it may be necessary to remove a particular enzyme used in an initial restriction enzyme reaction prior to completing all restriction enzyme digestions; this requires a time-consuming purification of the subcloning intermediate. Ideal methods for the subcloning of DNA molecules would permit the rapid transfer of the target DNA molecule from one vector to another in vitro or in vivo without the need to rely upon restriction enzyme digestions.
The present invention provides reagents and methods which comprise a system for the rapid subcloning of nucleic acid sequences in vivo and in vitro without the need to use restriction enzymes.
The present invention provides a method for the recombination of nucleic acid constructs, comprising: providing a first nucleic acid construct comprising, in operable order, an origin of replication, a first sequence-specific recombinase target site, and a nucleic acid of interest, a second nucleic acid construct comprising, in operable order, an origin of replication, a regulatory element and a second sequence-specific recombinase target site adjacent to and downstream from the regulatory element, and a site-specific recombinase; contacting the first and the second nucleic acid constructs with the site-specific recombinase under conditions such that the first and second nucleic acid constructs are recombined to form a third nucleic acid construct, wherein the nucleic acid of interest is operably linked to the regulatory element. The present invention contemplates the use of any type of regulatory element. In some embodiments of the present invention, the regulatory element comprises a promoter element, a fusion peptide (e.g., an affinity domain), or an epitope tag. In preferred embodiments, the nucleic acid of interest comprises a gene.
In some embodiments, the first nucleic acid construct further comprises a selectable marker.- In other embodiments, the second nucleic acid construct further comprises a selectable marker. The present invention contemplates that the first and second nucleic acid constructs both comprise selectable markers. In preferred embodiments the selectable markers of the first and second nucleic acid constructs are different from one another. Selectable markers include, but are not limited to a kanamycin resistance gene, an ampicillin resistance gene, a tetracycline resistance gene, a chloramphenicol resistance gene, a streptomycin resistance gene, a spectinomycin resistance gene, the aadA gene, the "PHgr"X174 E gene, the strA gene, and the sacB gene.
In preferred embodiments, the first nucleic acid construct further comprises a prokaryotic termination sequence. Prokaryotic termination sequences include, but are en not limited to the T7 termination sequence. In other preferred embodiments, the first nucleic acid construct further comprises a eukaryotic polyadenylation sequence. Polyadenylation sequences include, but are not limited to, the bovine growth hormone polyadenylation sequence, the simian virus 40 polyadenylation sequence, and the Herpes Simplex virus thymidine kinase polyadenylation sequence. In yet other preferred embodiments, the first nucleic acid construct further comprises a conditional origin of replication.
In preferred embodiments of the present invention, the first and second sequence-specific recombinase target sites are selected from the group consisting of loxP, loxP2, loxP3, loxP23, loxP511, loxB, loxC2, loxL, loxR, loxxcex9486, loxxcex94117, frt, dif, loxH and att. The present invention contemplates that the first and second sequence-specific recombinase target sites may comprise the same sequence or may comprise different sequences.
In yet other embodiments of the present invention, the first nucleic acid construct further comprises a polylinker.
The present invention contemplates that the recombination methods can be used in vitro and in vivo. In some in vivo embodiments, the site-specific recombinase is provided by a host cell expressing the site-specific recombinase. In some in vivo methods, the contacting of the first and the second nucleic acid constructs with the site-specific recombinase comprises introducing the first and said second nucleic acid constructs into a host cell under conditions such that the third nucleic acid construct is capable of replicating in the host cell.
The present invention further provides methods for precise transfer of nucleic acid molecules by recombination. In some embodiments, the first nucleic acid construct further comprises a third sequence-specific recombinase target site and said second nucleic acid constructs further comprises a fourth sequence-specific recombinase target site. In preferred embodiments, the first sequence-specific recombinase and the third sequence-specific recombinase in the first nucleic acid construct are located on opposite sides of the nucleic acid of interest. It is contemplated that the first and third sequence-specific recombinase target sites are contiguous with, adjacent to, or distant from the nucleic acid of interest. In particularly preferred embodiments the third and fourth sequence-specific recombinase target sites are selected from the group consisting of RS sites and Res sites, although other target sites are contemplated by the present invention. In some embodiments of the this method of the present invention, the first nucleic acid construct further comprises a third sequence-specific recombinase target site and the second nucleic acid constructs further comprises a fourth sequence-specific recombinase target site, wherein the method further comprises providing a second site-specific recombinase and the step of contacting the third nucleic acid construct with the second site-specific recombinase under conditions such that the third nucleic acid construct is recombined to form a fourth and a fifth nucleic acid construct.
The present invention also provides a recombined nucleic acid construct prepared according to any of the above methods.
The present invention further provides a method for the recombination of nucleic acid constructs, comprising: providing a vector, a linear nucleic acid molecule comprising a sequence complementary to at least a portion of said vector, and an E. coli host cell, wherein said host cell comprises an endogenous recombination system, a loss of function rec mutation, a suppressor, and a loss of function endogenous restriction modification system mutation; and introducing the vector and the linear nucleic acid molecule into the host cell under conditions such that the linear nucleic acid molecule and the vector are recombined to form a recombinant nucleic acid construct. In preferred embodiments the loss of function rec mutation is selected from the group consisting of recBC and recD. In other preferred embodiments, the suppressor comprises sbc. In yet other preferred embodiments, the loss of function endogenous restriction modification system mutation comprises hsdR.
The present invention further provides a method for generating a nucleic acid fusion on the 3xe2x80x2 end of the nucleic acid of interest in the first nucleic acid construct from above, comprising: providing a tagged linear nucleic acid sample comprising a tag to be added to the 3xe2x80x2 end of the nucleic acid of interest, and a sequence complementary to a region of the first nucleic acid construct that is 3xe2x80x2 of the nucleic acid of interest; and a host cell capable of endogenous homologous recombination of complementary nucleic acid molecules; and introducing the tagged linear nucleic acid sample and the first nucleic acid construct into the host cell under conditions such that the tagged linear nucleic acid sample and the first nucleic acid construct are recombined to form a tagged nucleic acid construct.
The present invention further provides a method for the cloning of nucleic acid libraries, comprising: providing a plurality of first nucleic acid constructs comprising, in operable order, an origin of replication, a first sequence-specific recombinase target site, and a nucleic acid member from a nucleic acid library, a plurality of second nucleic acid construct comprising, in operable order, an origin of replication, a regulatory element and a second sequence-specific recombinase target site adjacent to and downstream from the regulatory element, and a site-specific recombinase; contacting the plurality of first and second nucleic acid constructs with the site-specific recombinase under conditions such that the plurality of first and second nucleic acid constructs are recombined to form a plurality of third nucleic acid constructs, wherein the nucleic acid members from the nucleic acid library are operably linked to the regulatory elements. The present invention further provides a nucleic acid library prepared according to the above method.
The present invention also provides a method for the directional cloning of a nucleic acid molecule, comprising: providing first and second portions of a regulatory element, a first nucleic acid molecule comprising the first portion of the regulatory element; and a second nucleic acid molecule comprising the second portion of the regulatory element; and combining the first and the second nucleic acid molecules to produce a third nucleic acid molecule under conditions whereby an intact regulatory element is produced from the combination of the first and the second portions of the regulatory element, wherein the presence of the intact regulatory element in the third nucleic acid molecule indicates a direction of cloning of the first nucleic acid molecule with respect to the second nucleic acid molecule.
The present invention also provides a method for the directional cloning of a nucleic acid molecule, comprising providing: the nucleic acid molecule to be cloned, a first primer comprising sequence complementary to the nucleic acid molecule, a second primer comprising sequence complementary to the nucleic acid molecule and sequence corresponding to a first portion of a lacO site, amplification means, and a target nucleic acid molecule comprising a second portion of the lacO site; amplifying the nucleic acid molecule with the first and second primers to produce a modified nucleic acid molecule comprising the first portion of a lacO site; and ligating the modified nucleic acid molecule into the target nucleic acid such that, when cloned in the desired direction, an intact lacO site is produced. In some embodiments, the method further comprises the step of detecting the intact lacO site. In particularly preferred embodiments, the target nucleic acid molecule comprises pUNI-30.
The present invention further provides a method for regulated recombination in host cells that constitutively express a recombinase, comprising: providing a host cell expressing a recombinase, a first nucleic acid construct comprising an origin of replication, a first site-specific recombinase site, a second site-specific recombinase site that differs in sequence from the first site-specific recombinase site such that the recombinase will not initiate recombination between the first and second site-specific recombinase sites, and a selectable marker gene between the first and second site-specific recombinase sites, and a second nucleic acid construct comprising an origin of replication, a third site-specific recombinase target site, and a fourth site-specific recombinase target site that differs in sequence from the third site-specific recombinase site such that the recombinase will not initiate recombination between the third and fourth site-specific recombinase sites; and introducing the first and second nucleic acid constructs into the host cell under conditions such that the first and second nucleic acid constructs are recombined. In some embodiments, the method further comprises the step of selecting for a desired recombinant nucleic acid molecule using the selectable marker. In preferred embodiments, the first nucleic acid construct is a Univector. In alternative preferred embodiments, the second nucleic acid construct is a Univector.
The present invention also provides, a nucleic acid construct comprising, in operable order: a conditional origin of replication; a sequence-specific recombinase target site having a 5xe2x80x2 and a 3xe2x80x2 end; and a unique restriction enzyme site, said restriction enzyme site located adjacent to the 3xe2x80x2 end of the sequence-specific recombinase target site. In some embodiments, the construct further comprises a prokaryotic termination sequence. In yet other embodiments, the construct further comprises a eukaryotic polyadenylation sequence. The present invention contemplates the use of any prokaryotic termination sequence and any eukaryotic polyadenylation sequence. In preferred embodiments, the construct fruitier comprises one or more selectable marker genes. Selectable marker genes include, but are not limited to the kanamycin resistance gene, the ampicillin resistance gene, the tetracycline resistance gene, the chloramphenicol resistance gene, the streptomycin resistance gene, the strA gene, and the sacB gene. In preferred embodiments, the sequence-specific recombinase target site is selected from the group consisting of loxP, loxP2, loxP3, loxP23, loxP511, loxB, loxC2, loxL, loxR, loxxcex9486, loxxcex94117, frt, dif, loxH and att.
In some embodiments the construct further comprises a gene of interest inserted into the unique restriction enzyme site. In particular embodiments, the construct has the nucleotide sequence set forth in SEQ ID NO: 1 (FIG. 26A). In other embodiments, the construct further comprises a second sequence-specific recombinase target site. In preferred embodiments, the second sequence-specific recombinase target site is selected from the group consisting of RS site and a Res site. In yet other embodiments, the construct further comprises a polylinker.
The present invention further provides a nucleic acid construct comprising in 5xe2x80x2 to 3xe2x80x2 operable order: an origin of replication; a promoter element having a 5xe2x80x2 and a 3 end; and a sequence-specific recombinase target site having a 5xe2x80x2 and a 3xe2x80x2 end. In some embodiments, the construct further comprises a selectable marker gene.
The present invention also provides a nucleic acid construct comprising in operable order: a promoter element having a 5xe2x80x2 and a 3xe2x80x2 end; a first sequence-specific recombinase target site having a 5xe2x80x2 and a 3xe2x80x2 end, wherein the 3xe2x80x2 end of the promoter element is located upstream of the 5xe2x80x2 end of the sequence-specific recombinase target site; a gene of interest joined to the 3xe2x80x2 end of the sequence-specific recombinase target site such that a functional translational reading frame is created; a conditional origin of replication; a first selectable marker gene; a second sequence-specific recombinase target site; and an origin of replication. In some embodiments, the construct further comprises a second selectable marker gene.
The present invention also provides a method for the recombination of nucleic acid constructs, comprising: providing a first nucleic acid construct comprising a loxH site, a second nucleic acid construct comprising a loxH site; and a site-specific recombinase; and contacting the first and the second nucleic acid constructs with the site-specific recombinase under conditions such that the first and second nucleic acid constructs are recombined. The present invention also provides a recombined nucleic acid construct prepared according to the above method.